This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cryptosporidium hominis is listed as a biodefense priority since cryptosporidiosis is an emerging infectious disease with no effective therapies. Antifolate inhibitors of dihydrofolate reductase (DHFR) have proven to be effective therapeutics against other parasitic protozoa, but are ineffective against C. hominis due, in part, to low binding affinities to the enzyme. An analysis of the protein:ligand interactions in two crystal structures of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from C. hominis reveals that the interactions of specific residues in the active site of C. hominis DHFR with inhibitors provide a possible structural basis for the observed antifolate resistance. A comparison with the structure of human DHFR reveals active site differences that may be exploited for the design of species-selective inhibitors. We are working with a new concept, structure-guided library design that merges ideas from combinatorial chemistry and structure-based drug design, to create best-in-class C. hominis DHFR inhibitors that are both potent and selective for the pathogenic organism. During this synchrotron run, we plan to collect data for the co-crystals of DHFR-TS bound to several inhibitors.